Lyme disease is a common tick-borne illness caused by the Gram-negative bacterium Borrelia burgdorferi. An outer membrane protein of Borrelia burgdorferi, P66, has been suggested as a possible target for Lyme disease treatments. However, a lack of structural information available for P66 has hindered attempts to design medications to target the protein. Therefore, this study attempted to find methods for expressing and purifying P66 in quantities that can be used for structural studies. It was found that by using the PelB signal sequence, His-tagged P66 could be directed to the outer membrane of Escherichia coli, as confirmed by an anti-His Western blot. Further attempts to optimize P66 expression in the outer membrane were made, pending verification via Western blotting. The ability to direct P66 to the outer membrane using the PelB signal sequence is a promising first step in determining the overall structure of P66, but further work is needed before P66 is ready for large-scale purification for structural studies.

DNA nanotechnology is ideally suited for numerous applications from the crystallization and solution of macromolecular structures to the targeted delivery of therapeutic molecules. The foundational goal of structural DNA nanotechnology was the development of a lattice to host proteins for crystal structure solution. To further progress towards this goal, 36 unique four-armed DNA junctions were designed and crystallized for eventual solution of their 3D structures. While most of these junctions produced macroscale crystals which diffracted successfully, several prevented crystallization. Previous results used a fixed isomer and subsequent investigations adopted an alternate isomer to investigate the impact of these small sequence changes on the stability and structural properties of these crystals. DNA nanotechnology has also shown promise for a variety biomedical applications. In particular, DNA origami has been demonstrated as a promising tool for targeted and efficient delivery of drugs and vaccines due to their programmability and addressability to suit a variety of therapeutic cargo and biological functions. To this end, a previously designed DNA barrel nanostructure with a unique multimerizable pegboard architecture has been constructed and characterized via TEM for later evaluation of its stability under biological conditions for use in the targeted delivery of cargo, including CRISPR-containing adeno-associated viruses (AAVs) and mRNA.

Apolipoprotein (ApoE) plays an important role in the transport of lipids in the brain for normal functioning. There are three different isoforms of ApoE which are coded for by three alleles (E2, E3, E4). Patients carrying at least one copy of ApoE E4 are known to be at higher risk for developing Alzheimer’s disease (AD) and earlier onset of symptoms. This is due to the buildup of amyloid plaques and neurofibrillary tangles of the brain from the accumulation of tau proteins, which are associated with the progression of Alzheimer’s disease. However, findings on ApoE E2 have shown that it may be a protective allele since it is linked to a decreased risk of formation of amyloid plaques and neurofibrillary tangles. To study this phenomenon within the context of a local population group, polymerase chain reaction and gel electrophoresis were conducted on extracted DNA samples. The principal goal in this research study was to genotype ApoE variants using single nucleotide polymorphism (SNP) specific primers, and polymerase chain reaction to analyze the frequency in the Tempe population to determine future healthcare needs.

CRISPR-Cas based DNA precision genome editing tools such as DNA Adenine Base Editors (ABEs) could remedy the majority of human genetic diseases caused by point mutations (aka Single Nucleotide Polymorphisms, SNPs). ABEs were designed by fusing CRISPR-Cas9 and DNA deaminating enzymes. Since there is no natural enzyme able to deaminate adenosine in DNA, the deaminase domain of ABE was evolved from an Escherichia coli tRNA deaminase, EcTadA. Initial rounds of directed evolution resulted in ABE7.10 enzyme (which contains two deaminases EcTadA and TadA7.10 fused to Cas9) which was further evolved to ABE8e containing a single TadA8e and Cas9. The original EcTadA as well as the evolved TadA8e where shown to form homodimers in solution. Although it was shown that tRNA binding pocket in EcTadA is composed by both monomers, the significance of TadA dimerization in either tRNA or DNA deamination has not been demonstrated. Here we explore the role of TadA dimerization on the DNA adenosine deamination activity of ABE8e. We hypothesize that the dimerization of TadA8e is more important for the DNA deamination than for the tRNA deamination. To explore this, I conducted a urea titration on ABE8e to disrupt TadA8e dimerization and performed single turnover kinetics assays to assess DNA deamination rate of ABE8e’s. Results showed that DNA deamination rate and efficiency of ABE8e was already impaired at 4M urea and completely lost at 7M. Unfortunately, CD measurements at the equivalent urea concentrations indicate that the loss of activity is due to the unfolding of ABE8e rather than the disruption of TadA8e’s dimerization.

There are limited methods and techniques to quantitatively assess protein content in single cells or small cell populations of tissues. The standard protein insulin was used to understand how potential changes in the preparation or co-crystallization process could improve sensitivity and limit of detection through matrix assisted laser desorption ionization (MALDI) mass spectrometry analysis in Bruker’s Microflex LRF using polydimethylsiloxane (PDMS) reservoirs. In addition, initial imaging tests were performed on Bruker’s RapifleX MALDI Tissuetyper to determine the instrument’s imaging capabilities on proteins of interest through the use of a single layer “Christmas tree” microfluidic device, with the aim of applying a similar approach to future tissue samples. Data on 2µM insulin determined that a 95% laser power in the Microflex corresponded to 12-15% laser power in the RapifleX. Based on the experiments with insulin, the process of mixing insulin and saturated ɑ-Cyano-4-hydroxycinnamic acid (HCCA) matrix solvent in a 1:1 ratio using 10mM sodium phosphate buffer under area analysis is most optimized with a limit of detection value of 110 nM. With this information, the future aim is to apply this method to a double layer Christmas tree device in order to hopefully quantitatively analyze and image protein content in single or small cell populations.

Oceanic life is facing the deleterious aftermath of coral bleaching. To reverse the damages introduced by anthropological means, it is imperative to study fundamental properties of corals. One way to do so is to understand the metabolic pathways and protein functions of corals that contribute to the resilience of coral reefs. Although genomic sequencing and structural modeling has yielded significant insights for well-studied organisms, more investigation must be conducted for corals. Better yet, quantifiable experiments are far more crucial to the understanding of corals. The objective is to clone, purify, and assess coral proteins from the cauliflower coral species known as Pocillopora damicornis. Presented here is the pipeline for how 3-D structural modeling can help support the experimental data from studying soluble proteins in corals. Using a multi-step selection approach, 25 coral genes were selected and retrieved from the genomic database. Using Escherischia coli and Homo sapiens homologues for sequence alignment, functional properties of each protein were predicted to aid in the production of structural models. Using D-SCRIPT, potential pairwise protein-protein interactions (PPI) were predicted amongst these 25 proteins, and further studied for identifying putative interfaces using the ClusPro server. 10 binding pockets were inferred for each pair of proteins. Standard cloning strategies were applied to express 4 coral proteins for purification and functional assays. 2 of the 4 proteins had visible bands on the Coomassie stained gel and were able to advance to the purification step. Both proteins exhibited a faint band at the expected migration distance for at least one of the elutions. Finally, PPI was carried out by mixing protein samples and running in a native gel, resulting in one potential pair of PPI.

Molecular engineering is an emerging field that aims to create functional devices for modular purposes, particularly bottom-up design of nano-assemblies using mechanical and chemical methods to perform complex tasks. In this study, we present a novel method for constructing an RNA clamp using circularized RNA and a broccoli aptamer for fluorescence sensing. By designing a circular RNA with the broccoli aptamer and a complementary DNA strand, we created a molecular clamp that can stabilize the aptamer. The broccoli aptamer displays enhanced fluorescence when bound to its ligand, DFHBI-1T. Upon induction with this small molecule, the clamp can exhibit or destroy fluorescence. We demonstrated that we could control the fluorescence of the RNA clamp by introducing different complementary DNA strands, which regulate the level of fluorescence. Additionally, we designed allosteric control by introducing new DNA strands, making the system reversible. We explored the use of mechanical tension to regulate RNA function by attaching a spring-like activity through the RNA clamp to two points on the RNA surface. By adjusting the stiffness of the spring, we could control the tension between the two points and induce reversible conformational changes, effectively turning RNA function on and off. Our approach offers a simple and versatile method for creating RNA clamps with various applications, including RNA detection, regulation, and future nanodevice design. Our findings highlight the crucial role of mechanical forces in regulating RNA function, paving the way for developing new strategies for RNA manipulation, and potentially advancing molecular engineering. Although the current work is ongoing, we provide current progress of both theoretical and experimental calculations based on our findings.

The purpose of this experiment is to deliver DNA origami barrels loaded with Cas13d-gRNA binary complexes to HPV-16 and HPV-18 cervical cancer lines to make the cancer mortal. The production of Cas 13d has proven successful with a concentration of ~ 1mg/mL, but the activity assay performed has not shown conclusive evidence of Cas13d and guide RNA binary complex formation or activity. Successful annealing of the DNA origami barrel has been quantified by an agarose gel, but further quantification by TEM is in progress. Overall, steady progress is being made towards the goal of targeting HPV16 E6/E7 pre-mRNA with CRISPR/Cas13d.

Alzheimer’s disease (AD) is a common neurodegenerative disorder affecting approximately 10% of people aged 65 and up and 30-50% over 85. In pathological AD representations, a way to recognize early onset AD is the increased levels of pro-NGF in BFCNs that come from the downregulation of NGF with age. Pro-NGF has a higher affinity for p75NTR, which binds and participates in the pro-NGF-p75NTR-sortilin complex sequentially cleaved by α- and γ-secretase. Pro-NGF triggers apoptosis through the cleavage of the intracellular membrane by γ-secretase. Since γ-secretase physically cleaves off the intramembrane portion that promotes TNF- and Fas-dependent apoptotic signaling pathways, it has a crucial role in AD and must be better understood. This research aims to understand better and visualize γ-secretase and its actions, specifically with its interactions with the substrate p75NTR in the RIP process. To analyze γ-secretase function, the proteins must be produced and analyzed through the protein expression protocol. During protein production, DNA, cell concentrations, and optical density measurements were difficult to produce due to the incompetency of e. coli cells (DH5α), contamination of the Sf9 insect cell culture, and decreased viability of aged insect cells. We identified the problems and improved the conditions for future project development.

Sulfur oxidation is a process that is seen a wide variety of places. One particular place is Yellowstone national park where an abundance of hot springs are present. These acidic and hot places are prime locations for sulfur oxidation to occur. At a very basic level this is thought of as Sulfur, oxygen, and water forming sulfate and hydrogen. Many other reactions occur when an organism performs these processes, and many enzymes are used for this. This paper aimed to create, balance, and analyze the reactions involved in the paper Sulfur Oxidation in the Acidophilic Autotrophic Acidithiobacillus spp. (Wang et al., 2019) Once these reactions were balanced thermodynamic properties were found to evaluate the Gibbs Free Energy of these reactions. This allowed for a unique energy-based view of how this web of reactions relate to each other.